Project Summary Microtubule-based machines are responsible for the determination of cell shape, intracellular transport of organelles, chromosome separation during mitosis, and beating of cilia and flagella. Defects in the function of the motor proteins that drive these machines result in changes in cell shape, misplacement of organelles, infertility, chronic respiratory disease, and a wide array of developmental defects. The long-term goal of the proposed research is to understand the mechanisms that regulate the assembly, targeting, and activity of the dynein family of motors. We have identified several conserved genes that are involved in the assembly and coordination of dynein motors. We will continue to capitalize on the highly ordered structural organization of the flagellar axoneme and the ease of genetic analysis in Chlamydomonas to further characterize these genes and gene products and identify interacting components that regulate dynein activity. Our specific aims are: (1) To characterize three axonemal complexes, BOP2, PF10 and MBO2, that coordinate the assembly and activity of the inner dynein arms to modify the ciliary waveform. We hypothesize that each complex functions as an adaptor to attach specific dynein isoforms to the 96 nm repeat and interconnect the inner dynein arms to other regulatory complexes. We will use proteomic and molecular strategies to analyze the complexes in vitro, genetic analysis and motility assays to test for interactions in vivo, and high-resolution structural methods to localize subunits in situ (2) To determine the functions and locations of DRC subunits in the nexin link. We hypothesize that four N-DRC subunits have regulatory domains that facilitate interactions with the B-tubule, the outer dynein arms, and/or the radial spoke/calmodulin spoke complex. We will screen for mutations in these N-DRC subunits to reveal their specific roles in motility. We will localize them in the nexin link by SNAP-tagging and high resolution cryo-electron tomography. We will also analyze the pathway of nexin link assembly in vivo. (3) To characterize components that regulate motor activity and flagellar assembly. We have generated fluorescently tagged IFT motor subunits that rescue the relevant null mutations, and we will use these reagents to probe the mechanisms that regulate the cellular levels of IFT motors, IFT motor activity, and their effects on flagellar assembly. In particular, we will focus on the mechanism by which FLA4 facilitates flagellar assembly. We hypothesize that FLA4 is a conserved cytoplasmic factor that regulates the assembly, stability, and/or targeting of kinesins involved in flagellar assembly. The studies will provide basic information about the organization of ciliary dyneins and kinesins and associated regulatory components in cilia and flagella. Given the critical roles played by motor proteins and cilia and flagella in a wide range of human diseases, the studies will also have important implications for the development of diagnostic and therapeutic strategies in the treatment of human disease.